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Transformation
Transformation is one of the ways bacteria exchange DNA with each other, the other two ways are conjugation and transduction. Scientists then discovered how to make transformation happen to our advantage and now it is used widely through-out genetics. Transformation is a very popular genetic method that involves the uptake of foreign DNA into a cell. The most common type of transformation is transformation into an Eshcerchia coli cell when looking at recombinant plasmids. E. coli is used because it is a model organism, so many things are known about the cell that the plasmid/DNA will be transforming into. A transformation is helpful in genetics because of its ability to study bacteria as well as grow and replicate recombinant plasmids. Making Competent Cells First thing you need to do to begin a transformation is to make competent cells. Competent cells are cells that have a weaken cell membrane and are better able to uptake exogenous DNA. There are two types of competent cells; chemically competent and electrocompetent cells. Chemically competent cells require you to treat the cells with calcium chloride, while electrocompetent cells use electroporation to create pores in the cell using electric pulses. Many companies, however, make all ready competent cells so you will not need to worry about this. Cells should be kept in a -80 degrees Celcius freezer to keep them happy. History Transformation was first created by Fredrick Griffith, a British bacteriologist, in 1928. He discovered this method by looking at a strain of Streptococcus pneaumoniae. He found out that this strain could be made virulent just by being around virulent strains. In the 1970's scientists were able to find a way to use this transformation method that occurs naturally in bacteria and have it occur in vivo for scientific purposes. In 1970 Mandel and Higa showed that its possible to artificially induce a cell, E. coli in this case, to take up DNA from bacteriophage in the presence of calcium chloride. In 1972, Cohen, Chang, and Hsu were able to prove that it's also possible to use calcium chloride to allow for the uptake of plasmid DNA. Using these two discoveries, Hanahan was able to provide the protocol that is used today for the transformation of E. coli ''cells that is used routinely today in laboratories. Using electroporation was discovered later in the late 1980's as a way to make cells competent. Protocol To begin you should transfer the cells, in this case ''E.coli '' cells straight from the freezer onto ice. This will continue to make the cells happy. Mix 1-5 uL of the DNA into 20-50 uL of the competent cells(1). Normally if you are transforming a DNA construct use 50uL of competent cells, while if transforming a DNA ligation use 100 uL of cells. (standford.edu) To mix do not vortex just lightly filck the tube. Let this mixture sit on ice for 10 minutes. Next, you will want to heat shock the cells by placing the mixture in a 42 degrees Celcius for 20 seconds. This will allow the competent cells to become porous and allow the DNA into the membrane. Then put these cells back onto ice to reduce damage to the ''E.coli ''cells. To select for the colonies add 1mL LB to the mixture and let sit at 37 degrees Celcius for 1 hour. Plate 100uL of this mixture on LB/Amp plates overnight and pick off the colonies that grow the next day. References Bacterial Transformation and Transfection." ''Genome. The University of Oklahoma, n.d. Web. ."How to Do a Bacterial Transformation." Addgene: Protocol - How to Do a Bacterial Transformation. Addgene, n.d. Web. .MFT. "Transformation Protocol Using Heat Shock." Standford. Standford University, n.d. Web. ."Transformation (genetics)." Wikipedia. Wikimedia Foundation, 21 Sept. 2013. Web. 2013. ."Transformation." Overview of Transformation. Life Technologies, 2013. Web. .